Increased activation of the MAPK signaling pathway can induce malignancies. Oncogenic mutation V600E in BRAF is found in almost 50% of melanoma tumors. In addition, substantial percentages of BRAFV600E mutants are found in colorectal and thyroid cancers, about 10% and 40%, respectively. FDA-approved RAF inhibitors for melanoma show remarkable responses in BRAFV600E patients, however, treatment leads almost invariably to acquired resistance. Similarly, colorectal and thyroid cancers with BRAFV600E are largely resistant to RAF inhibitor treatment. Several studies have demonstrated that intrinsic or inhibitor-induced BRAFV600E dimerization plays a key role in the resistance mechanism. In melanoma, BRAFV600E usually acts as an active monomer, however, in a significant subset of tumors active spliced BRAFV600E dimers mediate primary resistance. Likewise, in large portions of upstream RAS-activated cancers like colorectal and thyroid, BRAFV600E dimers are promoted and the above drugs are ineffective. In light of the clinical relevance of BRAFV600E dimers to drive resistance in several tumors, we are currently in pressing need to develop novel inhibitors that target potently and specifically active BRAF dimers. We recently demonstrated the correlation of structural and biochemical effects of RAF inhibitors with their clinical manifestations. Using this knowledge, we initiated a systematic quest for novel inhibitors that specifically recognize BRAFV600E dimers. We discovered that Ponatinib, an FDA-approved drug, is such an inhibitor. In extensive preliminary studies, we characterized the effect of Ponatinib in melanoma, colorectal and other cancers and obtained its co-crystal structure with BRAFV600E and BRAFWT. Remarkably, the BRAF/Ponatinib structures demonstrated a perfectly symmetrical BRAF dimer and an allosteric inhibitor-binding mode, unprecedented for any BRAF inhibitor to date. Our observations generate an exceptional opportunity for drug design towards next- generation allosteric BRAF inhibitors that specifically inhibit BRAF dimers. Based on these observations, we created a ponatinib-hybrid compound, determined its co-crystal structure and validated its biochemical and cellular activity. Our results demonstrate excellent potency and specificity for BRAFV600E dimers compared to BRAFV600E monomers and provide a solid basis for the development of such first-in-class allosteric BRAF inhibitors. In this proposal, we will 1) design and synthesize ponatinib-hybrid compounds that bind to the novel allosteric pocket of BRAF and display improved binding and specificity to BRAFV600E dimers and desirable ADME properties, 2) robustly validate and optimize the potency and specificity of allosteric BRAF inhibitors in biochemical, biophysical and cellular experiments and 3) investigate the cellular mechanism of action of improved allosteric BRAFV600E inhibitors and their therapeutic potential in mouse tumor models. This project will investigate a broadly unmet therapeutic opportunity, namely the pharmacological targeting of BRAFV600E- dimerization dependent tumors that are resistant to current treatments.